1. Field
The invention relates to methods and apparatus for separating cells having differing sedimentation velocities.
2. State of the Art
The term "sedimentation velocity" refers to the rate at which a particle suspended in a liquid will fall due to gravity or other force applied. A particle, such as a cell, will fall at a rate which is dependent upon the strength of the gravitational force; the size, shape, and density of the particle; and the density and viscosity of the liquid in which the particle is suspended.
Differences in the sedimentation velocities of dissimilar particles may be used to separate the various particles from one another.
The gravitational force, the density of the supporting liquid, and the viscosity of the supporting liquid will be constant with respect to all particles which are loaded onto the supporting liquid in the separator apparatus. Thus, the only variables are the size, shape, and density of the individual particles. When the particles to be separated are cells, the number of variables decreases still further since cells are essentially spherical in shape and the densities of various cells are usually similar. Thus, the sedimentation velocity of a cell is a function of the size of the cell; the larger the cell, the higher its sedimentation velocity.
In the past, cells hve been separated by filling a chamber with a gradient of Ficoll (trademark of Pharmacia Fine Chemicals of Piscatuay, N.J., for a polysucrose material) or other solution such as sucrose or a large molecular weight polymer or protein. One device used to separate cells consists of a rectangular vessel having a height significantly greater than either dimension of its base. The gradient liquid is added to this vessel by means of a tube lowered into the top of the vessel, and the tube is raised by means of a float as the gradient is introduced so that gradient is always introduced at the top surface of liquid in the vessel. After introduction of the gradient, the cell suspension is loaded and the top sealed off. During gradient introduction and cell loading, both the gradient and cell suspension are passed through a sieve to minimize mixing. Then the vessel is rotated 90.degree. so that the thickness of isodense layers of liquid and the thickness of the layer of cells substantially decrease. After the cells have been given time to separate due to their differences in sedimentation velocity, the vessel is returned to its original position, a tube that is attached to an inverted funnel is lowered into the vessel, and the gradient is drawn off from the top and collected as various fractions.
Another device used to separate cells by sedimentation velocity is disclosed in U.S. Pat. No. 3,709,361. This device employs a cylindrical sedimentation vessel mounted for rotation between a horizontal position and a tilted position and has inlet/outlet ports located in the cylinder portion adjacent to the flat top and bottom walls. These ports extend from the cylinder portion at an obtuse angle to the top and bottom walls, respectively, in order to reduce the amount of mixing of isodense layers of liquid contained in the sedimentation vessel.